HST3/HST4-dependent deacetylation of lysine 56 of histone H3 in silent chromatin

The composition of posttranslational modifications on newly synthesized histones must be altered upon their incorporation into chromatin. These changes are necessary to maintain the same gene expression state at individual chromosomal loci before and after DNA replication. We have examined how one modification that occurs on newly synthesized histone H3, acetylation of K56, influences gene expression at epigenetically regulated loci in Saccharomyces cerevisiae. H3 K56 is acetylated by Rtt109p before its incorporation into chromatin during S phase, and this modification is then removed by the NAD⁺-dependent deacetylases Hst3p and Hst4p during G2/M phase. We found silenced loci maintain H3 K56 in a hypoacetylated state, and the absence of this modification in rtt109 mutants was compatible with HM and telomeric silencing. In contrast, loss of HST3 and HST4 resulted in hyperacetylation of H3 K56 within silent loci and telomeric silencing defects, despite the continued presence of Sir2p throughout these loci. These silencing defects in hst3Δ hst4Δ mutants could be suppressed by deletion of RTT109. In contrast, overexpression of Sir2p could not restore silencing in hst3Δ hst4Δ mutants. Together, our findings argue that HST3 HST4 play critical roles in maintaining the hypoacetylated state of K56 on histone H3 within silent chromatin.     

EBP1 regulates Suv39H1 stability via the ubiquitin-proteasome system in neural development

ErbB3-binding protein 1 (EBP1) is a multifunctional protein associated with neural development. Loss of Ebp1 leads to upregulation of the gene silencing unit suppressor of variegation 3-9 homolog 1 (Suv39H1)/DNA (cytosine 5)-methyltransferase (DNMT1). EBP1 directly binds to the promoter region of DNMT1, repressing DNA methylation, and hence, promoting neural development. In the current study, we showed that EBP1 suppresses histone methyltransferase activity of Suv39H1 by promoting ubiquitin-proteasome system (UPS)-dependent degradation of Suv39H1. In addition, we showed that EBP1 directly interacts with Suv39H1, and this interaction is required for recruiting the E3 ligase MDM2 for Suv39H1 degradation. Thus, our findings suggest that EBP1 regulates UPS-dependent degradation of Suv39H1 to govern proper heterochromatin assembly during neural development.     

The Rtt109 histone acetyltransferase facilitates error-free replication to prevent CAG/CTG repeat contractions

Lysine 56 is acetylated on newly synthesized histone H3 in yeast, Drosophila and mammalian cells. All of the proteins involved in histone H3 lysine 56 (H3K56) acetylation are important for maintaining genome integrity. These include Rtt109, a histone acetyltransferase, responsible for acetylating H3K56, Asf1, a histone H3/H4 chaperone, and Hst3 and Hst4, histone deacetylases which remove the acetyl group from H3K56. Here we demonstrate a new role for Rtt109 and H3K56 acetylation in maintaining repetitive DNA sequences in Saccharomyces cerevisiae. We found that cells lacking RTT109 had a high level of CAG/CTG repeat contractions and a twofold increase in breakage at CAG/CTG repeats. In addition, repeat contractions were significantly increased in cells lacking ASF1 and in an hst3Δhst4Δ double mutant. Because the Rtt107/Rtt101 complex was previously shown to be recruited to stalled replication forks in an Rtt109-dependent manner, we tested whether this complex was involved. However, contractions in rtt109Δ cells were not due to an inability to recruit the Rtt107/Rtt101 complex to repeats, as absence of these proteins had no effect on repeat stability. On the other hand, Dnl4 and Rad51-dependent pathways did play a role in creating some of the repeat contractions in rtt109Δ cells. Our results show that H3K56 acetylation by Rtt109 is important for stabilizing DNA repeats, likely by facilitating proper nucleosome assembly at the replication fork to prevent DNA structure formation and subsequent slippage events or fork breakage.     

A Reversible Histone H3 Acetylation Cooperates with Mismatch Repair and Replicative Polymerases in Maintaining Genome Stability

Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.     

A Novel Approach for Studying Histone H1 Function in Vivo

In this report, we investigate the mechanisms that regulate Drosophila histone H1 expression and its association with chromatin in vivo. We show that histone H1 is subject to negative autoregulation and exploit this result to examine the effects of mutations of the main phosphorylation site of histone H1.     

Schizosaccharomyces pombe Hst4 functions in DNA damage response by regulating histone H3 K56 acetylation

The packaging of eukaryotic DNA into chromatin is likely to be crucial for the maintenance of genomic integrity. Histone acetylation and deacetylation, which alter chromatin accessibility, have been implicated in DNA damage tolerance. Here we show that Schizosaccharomyces pombe Hst4, a homolog of histone deacetylase Sir2, participates in S-phase-specific DNA damage tolerance. Hst4 was essential for the survival of cells exposed to the genotoxic agent methyl methanesulfonate (MMS) as well as for cells lacking components of the DNA damage checkpoint pathway. It was required for the deacetylation of histone H3 core domain residue lysine 56, since a strain with a point mutation of its catalytic domain was unable to deacetylate this residue in vivo. Hst4 regulated the acetylation of H3 K56 and was itself cell cycle regulated. We also show that MMS treatment resulted in increased acetylation of histone H3 lysine 56 in wild-type cells and hst4Delta mutants had constitutively elevated levels of histone H3 K56 acetylation. Interestingly, the level of expression of Hst4 decreased upon MMS treatment, suggesting that the cell regulates access to the site of DNA damage by changing the level of this protein. Furthermore, we find that the phenotypes of both K56Q and K56R mutants of histone H3 were similar to those of hst4Delta mutants, suggesting that proper regulation of histone acetylation is important for DNA integrity. We propose that Hst4 is a deacetylase involved in the restoration of chromatin structure following the S phase of cell cycle and DNA damage response.     

CTR9/PAF1c regulates molecular lineage identity,histone H3K36 trimethylation and genomic imprinting during preimplantation development

Genome-wide epigenetic reprogramming is required for successful preimplantation development. Inappropriate or deficient chromatin regulation can result in defective lineage specification and loss of genomic imprinting, compromising normal development. Here we report that two members of the RNA polymerase II associated factor, homolog (Saccharomyces cerevisiae) complex (PAF1 complex) components, Ctr9 and Rtf1, are required during mammalian preimplantation development. We demonstrate that Ctr9-deficient embryos fail to correctly specify lineages at the blastocyst stage. Expression of some lineage specific factors is markedly reduced in Ctr9 knockdown embryos, including Eomes, Elf5 and Sox2, while others are inappropriately expressed (Oct4, Nanog, Gata6, Fgf4 and Sox17). We also show that several imprinted genes (Mest, Peg3, Snrpn and Meg3) are aberrantly expressed although allele specific DNA methylation is not altered. We document a loss of histone H3 lysine 36 trimethylation (H3K36me3) in Ctr9-deficient embryos and confirm that knockdown of either Setd2 or Rtf1 results in similar phenotypes. These findings show that the PAF1 complex is required for mammalian development, likely through regulation of H3K36me3, and indicate functional conservation of the PAF1 complex from yeast to mammals in vivo.     

Radically different amyloid conformations dictate the seeding specificity of a chimeric Sup35 prion

PHF2 belongs to a class of α-ketoglutarate-Fe²⁺-dependent dioxygenases. PHF2 harbors a plant homeodomain (PHD) and a Jumonji domain. PHF2, via its PHD, binds Lys4-trimethylated histone 3 in submicromolar affinity and has been reported to have the demethylase activity of monomethylated lysine 9 of histone 3 in vivo. However, we did not detect demethylase activity for PHF2 Jumonji domain (with and without its linked PHD) in the context of histone peptides. We determined the crystal structures of PHF2 Jumonji domain in the absence and presence of additional exogenous metal ions. When Fe²⁺ or Ni²⁺ was added at a high concentration (50 mM) and allowed to soak in the preformed crystals, Fe²⁺ or Ni²⁺ was bound by six ligands in an octahedral coordination. The side chains of H249 and D251 and the two oxygen atoms of N-oxalylglycine (an analog of α-ketoglutarate) provide four coordinations in the equatorial plane, while the hydroxyl oxygen atom of Y321 and one water molecule provide the two axial coordinations as the fifth and sixth ligands, respectively. The metal binding site in PHF2 closely resembles the Fe²⁺ sites in other Jumonji domains examined, with one important difference—a tyrosine (Y321 of PHF2) replaces histidine as the fifth ligand. However, neither Y321H mutation nor high metal concentration renders PHF2 an active demethylase on histone peptides. Wild type and Y321H mutant bind Ni²⁺ with an approximately equal affinity of 50 μM. We propose that there must be other regulatory factors required for the enzymatic activity of PHF2 in vivo or that perhaps PHF2 acts on non-histone substrates. Furthermore, PHF2 shares significant sequence homology throughout the entire region, including the above-mentioned tyrosine at the corresponding iron-binding position, with that of Schizosaccharomyces pombe Epe1, which plays an essential role in heterochromatin function but has no known enzymatic activity.     

Nicotinamide treatment reduces the levels of histone H3K4 trimethylation in the promoter of the mper1 circadian clock gene and blocks the ability of dexamethasone to induce the acute response

Circadian rhythms, which measure time on a scale of 24 h, are generated by one of the most ubiquitous endogenous mechanisms, the circadian clock. SIRT1, a class III histone deacetylase, and PARP-1, a poly(ADP-ribose) polymerase, are two NAD⁺-dependent enzymes that have been shown to be involved in the regulation of the clock. Here we present evidence that the metabolite nicotinamide, an inhibitor of SIRT1, PARP-1 and mono(ADP-ribosyl) transferases, blocks the ability of dexamethasone to induce the acute response of the circadian clock gene, mper1, while it concomitantly reduces the levels of histone H3 trimethylation of lysine 4 (H3K4me3) in the mper1 promoter. Moreover, application of alternative inhibitors of SIRT1 and ADP-ribosylation did not lead to similar results. Therefore, inhibition of these enzymes does not seem to be the mode by which NAM exerts these effects. These results suggest the presence of a novel mechanism, not previously documented, by which NAM can alter gene expression levels via changes in the histone H3K4 trimethylation state.     

Substitution as a mechanism for genetic robustness: the duplicated deacetylases Hst1p and Sir2p in Saccharomyces cerevisiae

How duplicate genes provide genetic robustness remains an unresolved question. We have examined the duplicated histone deacetylases Sir2p and Hst1p in Saccharomyces cerevisiae and find that these paralogs with non-overlapping functions can provide genetic robustness against null mutations through a substitution mechanism. Hst1p is an NAD⁺-dependent histone deacetylase that acts with Sum1p to repress a subset of midsporulation genes. However, hst1Δ mutants show much weaker derepression of target loci than sum1Δ mutants. We show that this modest derepression of target loci in hst1Δ strains occurs in part because Sir2p substitutes for Hst1p. Sir2p contributes to repression of the midsporulation genes only in the absence of Hst1p and is recruited to target promoters by a physical interaction with the Sum1 complex. Furthermore, when Sir2p associates with the Sum1 complex, the complex continues to repress in a promoter-specific manner and does not spread. Our results imply that after the duplication, SIR2 and HST1 subfunctionalized. The single SIR2/HST1 gene from Kluyveromyces lactis, a closely related species that diverged prior to the duplication, can suppress an hst1Δ mutation in S. cerevisiae as well as interact with Sir4p in S. cerevisiae. In addition, the existence of two distinct protein interaction domains for the Sir and Sum1 complexes was revealed through the analysis of a chimeric Sir2–Hst1 molecule. Therefore, the ability of Sir2p to substitute for Hst1p probably results from a retained but reduced affinity for the Sum1 complex that is a consequence of subfunctionalization via the duplication, degeneration, and complementation mechanism. These results suggest that the evolutionary path of duplicate gene preservation may be an important indicator for the ability of duplicated genes to contribute to genetic robustness.     

CSR-1 RNAi pathway positively regulates histone expression in C. elegans

Endogenous small interfering RNAs (endo-siRNAs) have been discovered in many organisms, including mammals. In C. elegans, depletion of germline-enriched endo-siRNAs found in complex with the CSR-1 Argonaute protein causes sterility and defects in chromosome segregation in early embryos. We discovered that knockdown of either csr-1, the RNA-dependent RNA polymerase (RdRP) ego-1, or the dicer-related helicase drh-3, leads to defects in histone mRNA processing, resulting in severe depletion of core histone proteins. The maturation of replication-dependent histone mRNAs, unlike that of other mRNAs, requires processing of their 3′UTRs through an endonucleolytic cleavage guided by the U7 snRNA, which is lacking in C. elegans. We found that CSR-1-bound antisense endo-siRNAs match histone mRNAs and mRNA precursors. Consistently, we demonstrate that CSR-1 directly binds to histone mRNA in an ego-1-dependent manner using biotinylated 2′-O-methyl RNA oligonucleotides. Moreover, we demonstrate that increasing the dosage of histone genes rescues the lethality associated with depletion of CSR-1 and EGO-1. These results support a positive and direct effect of RNAi on histone gene expression.     

Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells

Recognition and repair of damaged DNA occurs within the context of chromatin. The key protein components of chromatin are histones, whose post-translational modifications control diverse chromatin functions. Here, we report our findings from a large-scale screen for DNA-damage-responsive histone modifications in human cells. We have identified specific phosphorylations and acetylations on histone H3 that decrease in response to DNA damage. Significantly, we find that DNA-damage-induced changes in H3S10p, H3S28p and H3.3S31p are a consequence of cell-cycle re-positioning rather than DNA damage per se. In contrast, H3K9Ac and H3K56Ac, a mark previously uncharacterized in human cells, are rapidly and reversibly reduced in response to DNA damage. Finally, we show that the histone acetyl-transferase GCN5/KAT2A acetylates H3K56 in vitro and in vivo. Collectively, our data indicate that though most histone modifications do not change appreciably after genotoxic stress, H3K9Ac and H3K56Ac are reduced in response to DNA damage in human cells.